*3.5. Trypanosomatids Can Use Around Fivefold More Origins than the Minimum Required to Complete Replication within the S-Phase Duration*

To investigate how many times more origins than the minimum (MO) the organisms analyzed can use, we calculated the ratio between the angular coefficient (a value) of linear equations (y = ax + b) of maximum origins used and MO, shown as the red and black lines, respectively, in Figure 2. Here, we defined maximum origins used as the origins estimated by DNA combing, represented by the red lines in Figure 2; see material and methods.

Using this reasoning, we can estimate that *S. pombe* uses, on average, 1.44 times more origins than the MO, while in *S. cerevisiae*, this ratio is 2.12. Interestingly, in trypanosomatids, this ratio is higher. In *T. brucei* this ratio is 4.91, in *L. major* is 5.1, and in *T. cruzi* this ratio is so high that tends to infinity since *T. cruzi* needs only one origin per chromosome to replicate its nuclear genome (MO = 1 for all chromosomes), i.e., the MO linear equation is y = 1, as shown in Figure 3.

**Figure 3.** Trypanosomatids use around fivefold more origins than the minimum required. Angular coefficient (a-value) ratios between origins estimated by DNA combing and the minimum origins (MO) for *T. cruzi* (gray bar), *L. major* (orange bar), *T. brucei* (green bar), *S. cerevisiae* (blue bar), and *S. pombe* (purple bar).

− − − Although we cannot classify the total origins used as constitutive or non-constitutive, one question can be raised: what makes trypanosomatids apparently use a pool of origins much higher than the MO when compared to the yeasts *S. cerevisiae* and *S. pombe*? One possible explanation is that in trypanosomatids, unlike other eukaryotes, the majority of their genes are organized into large polycistronic clusters, which could favor replication stress through replication–transcription conflicts [8]. Replication stress, as reported in some studies [48,62], is a potential contributor for the activation of replication origins. However, although proposed by our group [8], this hypothesis needs to support more experimental assays to gain credibility. Another possibility is that the replication rate of *S. cerevisiae* and *S. pombe* are lower than those in trypanosomatids (1.6 kb·min−<sup>1</sup> in *S. cerevisiae*, 0.91 kb·min−<sup>1</sup> in *S. pombe*, and 2–3 kb·min−<sup>1</sup> in trypanosomatids), as shown in Table 1. *S. cerevisiae* has a chromosomes size and an S-phase duration similar to those found in trypanosomatids shown in Table 1 and Figure 1B. Thus, the only way to maintain robustness in the S-phase duration is by activating more origins. Apparently, *S. cerevisiae* does just that, but further studies are necessary to figure out its exact

dynamics of origin usage during the S-phase. On the other hand, *S. pombe* has larger chromosomes and a relatively short S-phase duration when compared to trypanosomatids, as displayed in Table 1 and Figure 1B. Moreover, as already mentioned, *S. pombe* does not have a robust S-phase [25,26] and its origins fire stochastically [50], which precludes any speculation regarding its peculiar dynamics of origin usage. However, unlike trypanosomatids, *S. pombe* appears to use a number of origins very close to the minimum required.

This is the first time a comparative analysis of the minimum number of origins has been successfully applied. These data may provide new insight into the understanding of origin usage during the S-phase and a new methodological framework for studying single-celled eukaryotes genomes.
